We identified a number of cell lines which can be transfected with RNA transcribed in vitro from our infectious cDNA clone and showed that the genomes replicate and that infectious virus is produced. HEV replicons expressing green fluorescent protein or luciferase were used to compare the effects of mutations in a non-coding region on replication in cell culture and in rhesus macaques. We showed that a viral protein that is phosphorylated in vitro is not required for genomic replication in cell culture but is required for infection of macaques, although it need not be phosphorylated. We developed a cell culture system which permits limited infection by wild-type HEV and used it to study neutralization and to show that HEV is more heat labile than is hepatitis A virus. We showed that a cDNA clone of swine HEV developed by a former post-doctoral student can replicate in our cell culture systems. We studied the humoral immune response to the HEV recombinant vaccine we developed previously and mapped the neutralization site, demonstrated that the vaccine induces antibodies that cross-react with viruses from all four genotypes, and showed that the vaccine contains all the immunodominant epitopes located in the capsid protein.